The human papillomaviruses (HPV) are major etiologic agents of several human cancers and have been found in >95% of cervical cancers, >50% of other anogenital cancers, and 20% of oral, laryngeal, and nasal cancers. Cervical cancers are associated with a high risk subset of HPVs of which HPV-16 is the most common. Progression of HPV-16 infection to high grade lesions and invasive cervical cancer requires significantly long lag periods, suggesting that multiple genetic events are required for malignant progression. The goal of this project is to identify changes in cellular and papillomaviral gene expression in cervical epithelia during progression of low grade squamous intraepithelial lesions (LGSIL) to high grade SIL (HGSIL) to invasive cervical cancer. Our initial studies have involved analysis of the Fragile Histidine Triad (FHIT) gene which is a candidate tumor suppressor gene located at 3p14.2, a locus that shows loss of heterozygosity (LOH) in cervical cancers. In addition, FHIT expression is absent in many epithelial malignancies including cervical cancer. This is frequently associated with the presence of aberrant FHIT transcripts. However, normal FHIT transcripts are sometimes found in cancer tissue and aberrant transcripts in normal tissue, raising some doubts about FHITs role as a tumor suppressor gene. It is not known if tumor cells express both normal and aberrant transcripts or if the normal transcripts are expressed in noncancer cells in the tumor tissue. We are using RT/PCR and immunohistochemistry to study FHIT expression in primary human foreskin keratinocytes, cervical cancer cell lines and invasive cervical cancers. Only normal FHIT transcripts were seen in normal keratinocytes, CaSki, and ME-180 cells. HT-3 cells showed both normal and aberrant transcripts while only aberrant transcripts were seen in SiHa and C33A cells. FHIT transcript was undetectable in HeLa cells. Absence of normal FHIT transcripts by nested RT/PCR correlated with absence of FHIT protein on Western blot and immunofluorescence. Frozen sections from 18 cervical cancers were analyzed for FHIT expression using nested RT/PCR. Normal FHIT transcript was found in 2 normal cervical specimens and one myometrial specimen. The majority of cancers showed both normal and aberrant transcripts, although a few tumors had only normal or aberrant transcripts. We are currently using manual and laser capture microdissection to determine if the normal transcripts in the cancers are expressed only in the contaminating stroma and normal epithelium. Five aberrant transcripts have been cloned and sequenced. All contain deletions of whole exons, including exon 5 which contains the translation initiation codon. Two transcripts also contain additional alternative exons that are flanked in the genome by 3 and 5 splice sites. It is not known if the aberrant transcripts arise through alternative splicing resulting from changes in splicing factors or are due to mutations in splice sites. We are currently investigating this. We are currently generating cDNA libraries from invasive cervical cancer cells obtained by Laser Capture Microdissection. Laser capture microdissection is used to avoid contamination with normal epithelia and stroma. These libraries will be used to identify novel cervix- specific genes and to give a preliminary expression profile for these cancers. Future analyses will utilize microarrays to profile gene expression in cervical cancer. In addition, we will use quantitative real time PCR to look at changes in alternative splicing during cervical carcinogenesis. January 1, 1999 to September 30, 1999 - Papillomaviruses, cervical cancer, post-transcriptional regulation, splicing, FHIT, gene expression, gene discovery, HPV, malignant conversion, Tumor suppressor genes, - Human Tissues, Fluids, Cells, etc.